Novel Intermediate Filament Proteins in Ramazzottius varieornatus and Todarodes pacificus

Abstract

Tardigrades are an extremophile invertebrate phylum increasingly popular in astrobiological research and science journalism on account of their extreme resilience. Ramazzottius varieornatus possesses three intermediate filament proteins; orthologs of an A-type lamin 2, a B-type lamin 1 and a cytoplasmic intermediate filament called cytotardin found in H. dujardini. This project aims to investigate to what extent the molecular similarities between the novel cytoplasmic intermediate filament and its lamin counterparts leads to similar in vitro properties. To this end, this protein, mutants thereof and a novel eye lens intermediate filament protein in Todarodes pacificus possessing molecular structural similarities to the tardigrade proteins, were produced and examined. Sequence and simulated biochemical analysis, as well as the nature of the rapid in vitro assembly of multiple superstructures indicates the lamin-like behaviour of the tardigrade cytoplasmic intermediate filament, whose central rod domain has the proportions of a Type V intermediate filament. Cytotardin is shown by electron microscopy, quantified by sedimentation assay, to be able to form paracrystalline structures across a wide range of pH and ionic strengths with a 24-25nm banding pattern characteristic of lamins. Substitution of an α-helix arginine residue for cysteine in the conserved LNTR motif mimics the keratin-14 mutation first associated with Epidermolysis Bullosa, causing partial loss of function. Deletion of the tail segment of cytotardin removes the ability to form sustainable paracrystals, let alone do so without divalent cations, supporting current models for intermediate filament assembly. Cytotardin and the A-type lamin appear closely related, based on their sequence identity and Western blot analysis and are capable of forming complex paracrystals with mobile structures. The in vitro networks of cytotardin and the A-Type lamin create viscous reticuli and gels in the solutions they inhabit. Western blot analysis shows both cytotardin and to a lesser extent the two R. varieornatus lamins are recognised by rabbit serum antibodies against T. pacificus eye lens protein.